# Unnatural Amino Acid Chemistry for Lysine Methyltransferase Substrate Discovery

> **NIH NIH R21** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2020 · $195,938

## Abstract

Project Abstract
Protein lysine methylation represents a prominent post-translational modification in biology. This modification
occurs in a multitude of proteins, including histones, transcription factors, chromatin modifying enzymes,
ribosomal proteins, cytoplasmic signaling enzymes, chaperones, spliceosomal factors, and cytoskeletal proteins.
Lysine methylation frequently modulates protein:protein interactions, often through the recruitment of
methyllysine binding factors, and has been implicated in regulating a diverse array of biological phenomena,
such as transcription, translation, DNA damage response, signal transduction, and protein chaperone function.
These modifications are catalyzed by S-adenosylmethionine (AdoMet)-dependent lysine methyltransferases
(KMTs), the majority of which belong to the SET domain family. The human genome encodes over 50 predicted
SET domain KMTs. It is fundamentally important to elucidate the substrate selectivities of these enzymes, as
methylation of their substrates defines their respective biological functions. Toward this goal, several techniques
have been developed to facilitate substrate identification of KMTs, including candidate-based approaches,
peptide and protein arrays, and a chemical affinity-mass spectrometry technique that utilizes AdoMet analogs
derivatized with bio-orthogonally reactive groups. Although these methods have aided in characterizing the
substrate selectivities of certain KMTs, substrate identification remains a persistent challenge. The Gozani
(Stanford University), Trievel (University of Michigan), Mehl (Oregon State University), and Larsen (University of
Michigan) laboratories have established an interdisciplinary collaboration to devise and implement a novel
method for discovering protein substrates of SET domain KMTs. This new approach is complementary to current
techniques and is based upon the introduction of an electrophilic unnatural amino acid (UAA) in the active sites
of KMTs. This UAA will facilitate proximity-induced chemical crosslinking with the lysine epsilon amino group in
protein substrates, with subsequent identification of the crosslinked substrates by mass spectrometry. We
envision that this methodology will enable systematic characterization of the substrate selectivities of SET
domain KMTs, yielding key insights into their biological functions and how dysregulation of these enzymes may
contribute to aberrant protein methylation and disease.

## Key facts

- **NIH application ID:** 10006583
- **Project number:** 5R21GM134285-02
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Or P. Gozani
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $195,938
- **Award type:** 5
- **Project period:** 2019-09-02 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10006583

## Citation

> US National Institutes of Health, RePORTER application 10006583, Unnatural Amino Acid Chemistry for Lysine Methyltransferase Substrate Discovery (5R21GM134285-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10006583. Licensed CC0.

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